Thrombopoietin (TPO) is a glycoprotein hormone involved regulation of platelet production. TPO promotes both the proliferation of megakaryocyte progenitors in the bone marrow and their maturation into platelet-producing megakaryocytes.
TPO has significant therapeutic value in the treatment of patients with reduced platelet count. In particular patients with many types of cancer suffer thrombocytopenias on account of myelosuppressive chemotherapy. Platelet transfusion has historically been the mainstay by which such patients have been supported. The availability of purified TPO from recombinant sources could enhance the options available for aggressive chemotherapy regimens and other patients at risk of bleeding complications due to their thrombocytopenia [Prow, D. & Vadhan-Raj, S. (1998) Oncology 12: 1597-1608].
At least two forms of recombinant human TPO have been developed for clinical trails. A truncated version comprising only the N-terminal 163 amino acids conjugated with polyethylene glycol is referred to as pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMDGF). A full length and glycosylated molecule is referred to as recombinant human thrombopoietin (rhTPO).
Both forms of TPO have been evaluated in Phase I/II trials, where they were given to cancer patients before receiving chemotherapy in order to boost platelet counts. The results of these trials have been reported [Basser R. L. et al (1996) Lancet; 348: 1279-1281; Basser R. L. et al (1997) Blood; 89: 3118-3128. Erratum in 1997; 90: 2513; Fannuchi M. et al, (1997), New England Journal of Medicine; 336: 404-409; Vadhan-Raj S. et al. (1997) Ann. Intern. Med; 126: 673-681 and Vadhan-Raj S. (1998) Semin. Hematol; 35: 261-268].
Both forms of TPO have been found to be immunogenic in a small proportion of subjects, and neutralising antibodies have also been demonstrated to both molecules [Hardy L, et al (1997) The Toxicologist; 36: 277; Li J, et al (2001) Blood; 98: 3241-3248; Koren E. (2002) Dev Biol (Basel); 109: 87-95; Basser R. L. et al (2002), Blood; 99: 2599-2602 and Koren E. (2002) Current Pharmaceutical Biotechnology; 3:349-360].
Clinical trials of PEG-rHuMDGF were abandoned in 1998 as neutralising antibodies could bind to endogenous TPO causing some of the patients and normal volunteers involved in the trials to become platelet transfusion dependent for several years. [Neumann T. A & Foote M. (2000) Cytokines Cell Mol Ther.: 6; 47-56].
Clearly for these subjects, an immune response has been mounted to the therapeutic TPO despite the fact that TPO is normally present in the circulation. The pivotal feature leading to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC class II molecules. Such peptide sequences are “T-cell epitopes” and are commonly defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Implicitly, a “T-cell epitope” means an epitope which when bound to MHC molecules can be recognised by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response. Patients who develop antibodies to TPO possess T cells that are capable of recognising peptide fragments of TPO bound to MHC class II molecules in their T cell repertoire.
To date no form of TPO has received regulatory approval as a therapeutic compound. From the foregoing there is clearly a continued need for TPO analogues with enhanced properties. There is a particular need for enhancement of the in vivo characteristics when administered to the human subject. In this regard, it is highly desired to provide TPO with reduced or absent potential to induce an immune response and enhanced biological potency in the human subject.
Others have provided TPO molecules and analogues [U.S. Pat. Nos. 5,989,538; U.S. Pat. No. 6,083,913; 5,879,673] including chemically modified and truncated forms [U.S. Pat. No. 5,989,538] and TPO fusion proteins [U.S. Pat. No. 6,066,318].
Koren et al [US Patent Application 20030077756] have identified peptide sequences in the C-terminal domain of human TPO that are able to interact with anti-TPO antibodies.
None of these teachings recognise the importance of T cell epitopes to the immunogenic properties of the protein nor have been conceived to directly influence said properties in a specific and controlled way according to the scheme of the present invention.
WO 03/104263 describes methods for the identification of CD4+ T-cell epitopes in cytokines including TPO. According to this method an epitope at residues 154-171 was defined. However, substitutions leading to a desired altered immunogenic response were suggested at residues outside of the epitope at residues 138, 139 and 140.
The co-owned application WO 02/068469 describes the results of an analysis of the entire TPO sequence for the presence of potential MHC class II binding ligands. The analysis therein is conducted using a computer simulation of the peptide MHC binding interaction. WO 02/068469 also provides multiple amino acid substitutions for achieving the disruption of the said potential epitope sequences.
The present invention is concerned also with TPO molecules in which amino acid substitution and or combinations of substitution have been conducted. In the present case, the molecules of the invention are fusion proteins comprising a human immunoglobulin constant region moiety linked with a human TPO mutein. Linkage to the immunoglobulin constant region domain causes the protein to become dimeric and these molecules additionally show increased potency.
This structure together with substitutions and combinations of substitutions in the TPO component confer the property of enhancing the biological activity of the molecule and also achieve a reduced immunogenic profile for the protein.
The general category of “human Fc fusion proteins” of which the present molecules are examples have been described previously [U.S. Pat. Nos. 5,541,087; 5,726,044 Lo et al (1998), Protein Engineering 11:495-500].